EP1799230B1 - Purines et pyrimidines inhibant des CDK et leur utilisation pour le traitement des maladies autoimmunes - Google Patents

Purines et pyrimidines inhibant des CDK et leur utilisation pour le traitement des maladies autoimmunes Download PDF

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EP1799230B1
EP1799230B1 EP05775408A EP05775408A EP1799230B1 EP 1799230 B1 EP1799230 B1 EP 1799230B1 EP 05775408 A EP05775408 A EP 05775408A EP 05775408 A EP05775408 A EP 05775408A EP 1799230 B1 EP1799230 B1 EP 1799230B1
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cdk2
cdk9
cdk7
pharmaceutically acceptable
inhibitor
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EP1799230A2 (fr
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Ariela Mario Negri Institute BENIGNI
Carla Mario Negri Institute ZOJA
Giuseppe Mario Negri Institute REMUZZI
Athos C/o Cyclacel Limited GIANNELLA-BORRADORI
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Cyclacel Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to compounds for use in a method of treating diseases associated with antinuclear antibodies. More specifically, but not exclusively, the invention relates to compounds for use in methods of treating autoimmune rheumatic diseases such as human systemic lupus erythematosus (SLE), and pharmaceutical compositions and combinations therefor.
  • autoimmune rheumatic diseases such as human systemic lupus erythematosus (SLE)
  • SLE systemic lupus erythematosus
  • the purpose of the immune system is to protect the body from potentially harmful substances (antigens) such as microorganisms, toxins, cancer cells, and foreign blood or tissues from another person or species.
  • Antigens are destroyed by the immune response, which includes production of antibodies (molecules that attach to the antigen and make it more susceptible to destruction) and sensitized lymphocytes (specialized white blood cells that recognize and destroy particular antigens).
  • Immune system disorders occur when the immune response is inappropriate, excessive, or lacking.
  • Autoimmune disorders refers to any disease characterized by abnormal functioning of the immune system that causes the immune system to produce antibodies against its own tissues. This is caused by a hypersensitivity reaction where the immune system reacts to substances that it normally would ignore, i.e. normal "self" body tissues.
  • the immune system is capable of differentiating "self” from “non-self” tissue.
  • Some immune system cells lymphocytes
  • lymphocytes become sensitized against “self” tissue cells, but these faulty lymphocytes are usually controlled (suppressed) by other lymphocytes.
  • Autoimmune disorders occur when the normal control process is disrupted, or if normal body tissue is altered so that it is no longer recognized as “self.”
  • autoimmune disorders typically result in destruction of one or more types of body tissues, abnormal growth of an organ, or changes in organ function.
  • the disorder may affect only one organ or tissue type or may affect multiple organs and tissues.
  • Organs and tissues commonly affected by autoimmune disorders include blood components such as red blood cells, blood vessels, connective tissues, endocrine glands such as the thyroid or pancreas, organs such as the kidney or liver, muscles, joints, and skin.
  • autoimmune or autoimmune-related disorders include: Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, drug-induced lupus erythematosus, multiple scleroris, myasthenia gravis, Reiter's syndrome and Grave's disease.
  • autoimmune diseases vary widely depending on the type of disease. However, autoimmune diseases are often associated with non-specific symptoms such as fatigue, dizziness, malaise (non-specific feeling of not being well), fever, and low-grade temperature elevations.
  • Specific autoimmune disease results in the destruction of an organ or tissue resulting in decreased functioning of an organ or tissue (for example, the islet cells of the pancreas are destroyed in diabetes), and/or an increase in size of an organ or tissue (for example, thyroid enlargement in Grave's disease). Symptoms vary depending on the specific disorder and the organ or tissue affected.
  • Autoimmunity is controlled through balanced suppression of the immune system.
  • the aim is to reduce the immune response against normal body tissue whilst leaving intact the immune response against micro-organisms and abnormal tissues.
  • Clinical treatments for autoimmune diseases typically involve the use of corticosteroids and immunosuppressants (including cyclophosphamide or azathioprine) which reduce the immune response.
  • corticosteroids including cyclophosphamide or azathioprine
  • the present invention seeks to provide alternative compounds for use in therapeutic methods for treating autoimmune diseases which ideally are capable of reducing symptoms and controlling the autoimmune process, whilst maintaining the ability to fight disease. More particularly, the invention relates to compounds for use in the treatment of diseases associated with antinuclear antibodies, especially autoimmune diseases such as human systemic lupus erythematosus. The invention also seeks to provide pharmaceutical combinations and compositions suitable for treating such disorders.
  • a first aspect of the invention relates to use of a purine inhibitor of CDK2 and/or CDK7 and/or CDK9 selected from or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating drug-induced lupus or systemic lupus erythematosis (SLE).
  • SLE systemic lupus erythematosis
  • a second aspect of the invention relates to a combination comprising purine inhibitor of CDK2 and/or CDK7 and/or CDK9, selected from: or a pharmaceutically acceptable salt thereof, and methylprednisolone.
  • a third aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the above combination and a pharmaceutically acceptable carrier, diluent or excipient.
  • a fourth aspect of the invention relates to a pharmaceutical product comprising a purine inhibitor of CDK2 and/or CDK7 and/or CDK9 selected from or a pharmaceutically acceptable salt thereof, and methylprednisolone as a combined preparation for simultaneous, sequential or separate use in therapy.
  • a fifth aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising:
  • a sixth aspect of the invention relates to the use of a purine inhibitor of CDK2 and/or CDK7 and/or CDK9 selected from or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating drug-induced lupus, or systemic lupus erythematosis (SLE), wherein the medicament is for use in combination with methylprednisolone.
  • a purine inhibitor of CDK2 and/or CDK7 and/or CDK9 selected from or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating drug-induced lupus, or systemic lupus erythematosis (SLE), wherein the medicament is for use in combination with methylprednisolone.
  • a seventh aspect of the invention relates to the use of methylprednisolone in the preparation of a medicament for treating drug-induced lupus, or systemic lupus erythematosis (SLE), wherein the medicament is for use in combination with a purine inhibitor of CDK2 and/or CDK7 and/or CDK9 selected from or a pharmaceutically acceptable salt thereof.
  • SLE systemic lupus erythematosis
  • An eighth aspect of the invention relates to the use of a purine inhibitor of CDK2 and/or CDK7 and/or CDK9 selected from or a pharmaceutically acceptable salt thereof, and methylprednisolone, in the preparation of a medicament for treating drug-induced lupus, or systemic lupus erythematosis (SLE).
  • a purine inhibitor of CDK2 and/or CDK7 and/or CDK9 selected from or a pharmaceutically acceptable salt thereof, and methylprednisolone
  • a ninth aspect relates to the use of a purine inhibitor of CDK2 and/or CDK7 and/or CDK9 selected from or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of drug-induced lupus, or systemic lupus erythematosis (SLE), wherein said treatment comprises administering to a subject simultaneously, sequentially or separately the purine inhibitor of CDK2 and/or CDK7 and/or CDK9, or a pharmaceutically acceptable salt thereof, and methylprednisolone.
  • SLE systemic lupus erythematosis
  • a tenth aspect of the invention relates to a purine inhibitor of CDK2 and/or CDK7 and/or CDK9 selected from or a pharmaceutically acceptable salt thereof, for use in treating drug-induced lupus or systemic lupus erythematosis.
  • An eleventh aspect of the invention relates to purine inhibitor of CDK2 and/or CDK7 and/or CDK9 selected from or a pharmaceutically acceptable salt thereof and methylprednisolone, for use in treating drug-induced lupus or systemic lupus erythematosis.
  • the present invention relates to the use of one or more purine inhibitors of CDK2 and/or CDK7 and/or CDK9 selected from those set out above.
  • the inhibitor may be an inhibitor of any one of CDK2, CDK7 or CDK9, or any combination thereof.
  • the inhibitor is an inhibitor of CDK2.
  • the inhibitor is an inhibitor of CDK7.
  • the inhibitor is an inhibitor of CDK9.
  • the inhibitor is an inhibitor of CDK7 and CDK9.
  • the inhibitor exhibits an IC 50 value for one or more of CDK2, CDK7 or CDK9 of less than 1 mM, more preferably less than 100 ⁇ M, more preferably still, less than 50 ⁇ M. more preferably less than 25 ⁇ M, more preferably less than 10 ⁇ M, even more preferably less than 1 ⁇ M or 0.5 or 0.1 ⁇ M.
  • CDK2 and/or CDK7 and/or CDK9 are disclosed in EP 0874847B (CNRS), WO 03/002565 (Cyclacel Limited), WO 04/016613 (Cyclacel Limited) and WO 04/016612 (Cyclacel Limited).
  • the inhibitor of CDK2 and/or CDK7 and/or CDK9 is selected from the following: and pharmaceutically acceptable salts thereof.
  • the inhibitor of CDK2 and/or CDK7 and/or CDK9 is roscovitine.
  • Roscovitine or 2-[(1-ethyl-2-hydroxyethyl)amino]-6-benzylamine-9-isopropylpurine is also described as 2-(1-D,L-hydroxymethylpropylamino)-6-benzylamine-9-isopropyl- purine.
  • Roscovitine encompasses the resolved R and S enantiomers, mixtures thereof, and the racemate thereof.
  • CYC202 refers to the R enantiomer of roscovitine, namely. 2-(1-R-hydroxymethylpropylamino)-6-benzylamino-9-isopropylpurine, the structure of which is shown below.
  • the in vitro activity of roscovitine is as follows: Kinase IC 50 ( ⁇ M) Cdk1/cyclin B 2.7 Cdk2/cyclin A 0.7 Cdk2/cyclin E 0.1 Cdk7/cyclin H 0.5 Cdk9/cyclin T1 0.8 Cdk4/cyclin D1 14.2 ERK-2 1.2 PKA >50 PKC >50
  • roscovitine in the treatment of autoimmune disorders is documented in the art, to date, there has been no suggestion that it would be effective in reducing the level of antinuclear antibodies. Nor has there been any teaching or suggestion in the art that roscovitine could be used in combination therapy with methylprednisolone in the treatment of autoimmune disorders.
  • the roscovitine is in the form of the R enantiomer, namely 2-(1-R-hydroxymethylpropylamino)-6-benzylamino-9-isopropyl- purine, hereinafter referred to as "CYC202".
  • the present invention relates to the use the above described inhibitors of CDK2 and/or CDK7 and/or CDK9, or pharmaceutically acceptable salts thereof, in the preparation of a medicament for treating drug-induced lupus or SLE.
  • the inhibitor of CDK2 and/or CDK7 and/or CDK9, or pharmaceutically acceptable salt thereof is administered in an amount sufficient to down-regulate the levels of antinuclear antibodies.
  • Antinuclear antibodies are unusual antibodies, detectable in the blood, that have the capability of binding to certain structures within the nucleus of the cells. ANAs are found in patients whose immune system may be predisposed to cause inflammation against their own body tissues. Antibodies that are directed against one's own tissues are referred to as autoantibodies. The propensity for the immune system to work against its own body is referred to as autoimmunity.
  • Antinuclear antibodies are a diverse group of antibodies, often directed to large cellular complexes containing protein and nucleic acid components. The most frequently occurring antinuclear antibodies react with components of DNA-protein or RNA-protein complexes [Van Venrooij W.J. et al; Von Mulen C. A. et al]. Studies have indicated that the production of these autoantibodies, which are generally high-titre, high affinity IgG antibodies is T-cell dependent and driven by the host autoantigen [Reichlin M. et al].
  • tissue damage For a disease to be defined as autoimmune, the tissue damage must be shown to be caused by an adaptive immune response to self antigens.
  • Autoimmune diseases can be mediated by autoantibodies and/or by auto-reactive T cells, and tissue damage can result from direct attack on the cells bearing the antigen, from immune-complex formation, or from local inflammation.
  • T cells can be involved directly in inflammation or cellular destruction, and they are also required to sustain autoantibody responses.
  • B cells may be important antigen-presenting cells for sustaining autoantigen-specific T-cell responses.
  • CD4 T cells selectively activate those B cells that bind epitopes that are physically linked to the peptide recognized by the T cell.
  • both autoreactive B cells and autoreactive T cells are required for a disease involving autoimmunity.
  • Anti-nuclear antibody production can only be studied in vivo since the production of these antibodies requires a dysfunctional immune system comprising both B and T cells, and a failure to select and destroy immune cells that recognise self.
  • T cell proliferation assay In vitro assays for T cell function are appropriate screening tools to identify compounds that may have the ability to modulate the immune response in the complex situation of autoimmune disease.
  • One such assay is a T cell proliferation assay, further details of which are set forth in the accompanying Examples.
  • autoimmune diseases involving anti-nuclear antibody production normal T cell function is required to stimulate autoantibody production by B cells. Accordingly, compounds affecting T cell function (one measure of T cell function is the ability to proliferate in response to an immune stimulus) should also prevent the formation of autoantibodies, by controlling the ability of T cells to communicate with B cells and in addition by preventing T cells from migrating to the site of auto-immune damage.
  • anti-DNA antibodies includes both anti-DNA antibodies, anti-RNA antibodies and antibodies directed against protein nuclear components.
  • Diseases associated with antinuclear antibodies include autoimmune rheumatic diseases and organ specific autoimmunity.
  • the autoimmune rheumatic disease is selected from drug induced lupus, systemic lupus erythematosis (SLE).
  • SLE systemic lupus erythematosis
  • the autoimmune rheumatic disease is systemic lupus erythematosis (SLE).
  • the invention relates to the treatment of systemic lupus erythematosus (also known as disseminated lupus erythematosus; SLE; lupus; lupus erythematosus) is a chronic, inflammatory autoimmune disorder that may affect many organ systems including the skin, joints and internal organs. Although people with the disease may have many different symptoms, some of the most common ones include extreme fatigue, painful or swollen joints (arthritis), unexplained fever, skin rashes, and kidney problems. At present there is no cure for SLE.
  • systemic lupus erythematosus also known as disseminated lupus erythematosus; SLE; lupus; lupus erythematosus
  • SLE disseminated lupus erythematosus
  • lupus lupus erythematosus
  • the disease affects nine times as many women as men and appears more common in people of African descent. It may occur at any age, but appears most often in people between the ages of 10 and 50 years. SLE may also be caused by certain drugs. When this occurs, it is known as drug-induced lupus erythematosus and it is usually reversible when the medication is stopped.
  • the course of the disease may vary from a mild episodic illness to a severe fatal disease. Symptoms also vary widely in a particular individual over time and are characterized by periods of remission and exacerbation. Some of the most common symptoms of lupus include painful or swollen joints (arthritis), unexplained fever, and extreme fatigue. Other symptoms of lupus include chest pain, hair loss, anemia (a decrease in red blood cells), mouth ulcers, and pale or purple fingers and toes from cold and stress. Some people also experience headaches, dizziness, depression, confusion, or seizures. New symptoms may continue to appear years after the initial diagnosis, and different symptoms can occur at different times. At its onset, only one organ system may be involved. Additional organs may become involved later.
  • Lupus is characterized by periods of illness, called flares, and periods of wellness, or remission.
  • flares periods of illness
  • the doctor will develop a treatment plan based on the patient's age, sex, health, symptoms, and lifestyle. In developing a treatment plan, the doctor has several goals: to prevent flares, to treat them when they do occur, and to minimize organ damage and complications.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • Antimalarials are another type of drug commonly used to treat lupus. Although they are generally used to treat fatigue, joint pain, skin rashes, and inflammation of the lungs, clinical studies have found that continuous treatment with antimalarials may prevent flares from recurring. Side effects of anti-malarials can include stomach upset and, extremely rarely, damage to the retina of the eye.
  • corticosteroid hormones work by rapidly suppressing inflammation and can be given by mouth, in creams applied to the skin, or by injection.
  • Short-term side effects of corticosteroids include swelling, increased appetite, and weight gain. These side effects generally stop when the drug is stopped. However, it can often be dangerous to stop taking corticosteroids suddenly, so there is a need to wean the patient off them over an extended period of time.
  • Long-term side effects of corticosteroids can include stretch marks on the skin, weakened or damaged bones (osteoporosis and osteonecrosis), high blood pressure, damage to the arteries, high blood sugar (diabetes), infections, and cataracts. Typically, the higher the dose and the longer they are taken, the greater the risk and severity of side effects.
  • immunosuppressives For patients whose kidneys or central nervous systems are affected by lupus, immunosuppressives may be used. Immunosuppressives restrain the overactive immune system by blocking the production of immune cells. Side effects may include nausea, vomiting, hair loss, bladder problems, decreased fertility, and increased risk of cancer and infection. The risk for side effects increases with the length of treatment. As with other treatments for lupus, there is a risk of relapse after the immunosuppressives have been stopped.
  • the present invention provides alternative therapeutic treatments for SLE and drug-induced lupus.
  • the invention aims to provide alternative treatments to immunosuppressants and steroids, which at least in the acute phase of the disease have excellent therapeutic effects but which lack major long term side effects.
  • NZB/W F1 hybrid mice spontaneously develop a severe autoimmune disease reminiscent of human systemic lupus erythematosus.
  • the disease manifests with early antinuclear antibody formation, development of an immune complex glomerulonephritis with proteinuria and progression to renal insufficiency with time, that also causes premature mortality in these mice.
  • It is a disease of T and B cell dysfunction with the formation of autoantibodies against nuclear and endogenous antigens, among which nucleosomes, DNA complexed to histones, seem to be the most prominent.
  • nucleosomes are generated by apoptosis, a process that appears disturbed in lupus.
  • nucleosomes act as autoantigens to drive a T cell-dependent immune response, whose critical components are nucleosome-specific antibodies and nucleosome/IgG complexes. These bind to intrinsic constituents of the glomerular basement membrane and promote inflammation.
  • Cytokines and chemoattractants generated in exuberant amount by renal resident and infiltrating cells amplify and perpetuate immune complex-mediated injury.
  • glomerular changes in NZB/W mice include endocapillary hypercellularity associated with focal extracapillary proliferation; immune-type deposits are detected in the mesangium and on subendothelial aspect of the glomerular basement membrane (GBM). Tubular damage, interstitial inflammation and fibrosis are severe.
  • GBM glomerular basement membrane
  • CDK2 positive cell cycle proteins
  • cyclins D, E, A positive cell cycle proteins
  • CDK2 protein levels and activity are increased in experimental mesangioproliferative glomerulonephritis (Thy nephritis) characterized by mesangial cell proliferation.
  • Thy nephritis mesangioproliferative glomerulonephritis characterized by mesangial cell proliferation.
  • inhibiting CDK2 decreased mesangial cell proliferation and matrix protein accumulation and improved renal function.
  • month 8 At the end of the study (month 8) while only four of thirteen NZB/W mice (31 %) that had been treated with vehicle were alive, ten of thirteen mice (77%) and ten of fourteen mice (71%) treated with 200 and 100 mg/kg CYC202, respectively, survived.
  • the percentage of survival was not different from that recorded in vehicle-treated mice.
  • the amount of the inhibitor of CDK2 and/or CDK7 and/or CDK9 is sufficient to delay the onset of proteinuria and renal function impairment.
  • CYC202 given as a preventive therapy from 2 months of age significantly delayed the onset of proteinuria compared to vehicle, in a dose-dependent manner (% proteinuric mice, month 8: 200 mg /kg, 23%, P ⁇ 0.01 vs vehicle; 100 mg/kg, 43%, P ⁇ 0.05 vs vehicle).
  • CYC202 was administered to lupus mice from 5 months of age a tendency toward a reduced percentage of proteinuric mice in respect to vehicle was observed, which however did not reach the statistical significance.
  • Renal function assessed by serum blood urea nitrogen (BUN), was measured at 5 and 8 months. At 5 months serum BUN levels were within the normal range ( ⁇ 29 mg/dl) in all the experimental groups. In the vehicle group, renal function deteriorated with time and at 8 months 50% of the surviving animals had BUN levels ⁇ 30 mg/dl. CYC202 given as preventive therapy from 2 months of age resulted in a better renal function of lupus mice, whereas it was not effective when administered at the later age of 5 months.
  • the amount of the inhibitor of CDK2 and/or CDK7 and/or CDK9 is sufficient to down-regulate the levels of anti-nuclear, specifically anti-DNA antibodies.
  • mice Elevated anti-DNA antibody levels are characteristic of NZB/W F1 mice. Mice given vehicle exhibited increasing levels of anti-DNA antibodies over time. Either at 5 or 8 months of age, mice treated from 2 months with CYC202 at both doses showed anti-DNA antibody levels significantly lower than vehicle. In the group of mice receiving CYC202 from 5 months anti-DNA antibody concentration was numerically, albeit not significantly lower than vehicle at 8 months.
  • the inhibitor of CDK2 and/or CDK7 and/or CDK9 is in an amount sufficient to reduce glomerular and tubointerstitial changes.
  • mice given vehicle revealed glomerular changes with intracapillary hypercellularity associated with a focal extracapillary proliferation.
  • Immune deposits were detected in the mesangium and on the subendothelial aspect of the glomerular basement membrane. Tubular damage and interstitial inflammation were also observed.
  • Treatment from 2 months of age with CYC202 markedly limited glomerular hypercellularity, immune deposits, and tubulointerstitial damage. These effects were more pronounced when CYC202 was given at the dose of 200 mg/kg. Only a mild effect on renal morphology was observed in mice administered CYC202 from 5 months of age.
  • the amount of the inhibitor of CDK2 and/or CDK7 and/or CDK9 is sufficient to inhibit T-cell proliferation induced by PMA and ConA.
  • the inhibitor of CDK2 and/or CDK7 and/or CDK9 is in an amount sufficient to down-regulate the expression of Mcl-1.
  • the amount of the inhibitor of CDK2 and/or CDK7 and/or CDK9 is in an amount sufficient to reduce the interstitial accumulation of mononuclear cells.
  • Kidneys were analysed for F4/80 positive monocytes/macrophages by immunohistochemical technique. A marked accumulation of F4/80 positive cells was present in the renal interstitium of NZB/W mice given vehicle. Preventive treatment from 2 months of age with 200 mg/kg CYC202 remarkably reduced the number of F4/80 positive monocytes/macrophages in respect to vehicle. CYC202 at the dose of 100 mg/kg limited albeit not to a statistically significant degree the interstitial accumulation of mononuclear cells. A numerical reduction of F4/80 positive cells was observed in the therapeutic study (treatment from 5 months of age).
  • CYC202 200 and 100 mg/kg given as a preventive therapy from 2 months of age, retarded renal manifestation of lupus in NZB/W mice and remarkably prolonged life as compared to animals given vehicle.
  • CYC202 delayed the onset of proteinuria and renal function impairment, and limited glomerular and tubulointerstitial changes including the interstitial accumulation of mononuclear cells, glomerular hypercellularity and immune deposits. The effects are more pronounced at the dose of 200 mg/kg.
  • a remarkable finding of the present study was the reduction of the levels of anti-DNA antibodies by CYC202, which could possibly be attributed to CYC202 effects on T cells, which in turn may affect B cells.
  • CYC202 is also known to have an effect on transcription, including downregulation of the anti-apoptotic protein mcl-1 and is thus capable of altering the balance of survival signals in cells.
  • Anergic T cells may be specifically sensitive to this mechanism of action.
  • CYC202 100 mg/kg
  • the administration of CYC202 initiated at 5 months of age resulted in a mild reduction of the percentage of proteinuric mice and of renal damage in respect to vehicle-mice. Survival was not ameliorated. The modest effect was probably due to the low dose.
  • the medicament is for use in combination therapy with methylprednisolone.
  • the term “combination therapy” refers to therapy in which the methylprednisolone and the inhibitor of CDK2 and/or CDK7 and/or CDK9, are administered, if not simultaneously, then sequentially within a timeframe that they both are available to act therapeutically within the same time-frame.
  • Another aspect of the invention relates to a combination comprising an inhibitor of CDK2 and/or CDK7 and/or CDK9 as described above, or a pharmaceutically acceptable salt thereof, and methylprednisolone.
  • the combination has a synergistic effect, i.e. the combination is synergistic.
  • Methylprednisolone is a synthetic (man-made) corticosteroid.
  • the chemical name is 11,17,21-trihydroxy-6-methyl-(6a,11b)-pregna-1,4-diene-3,20-dione.
  • Corticosteroids are naturally- occurring chemicals produced by the adrenal glands located adjacent to the kidneys. Corticosteroids block inflammation and are used in a wide variety of inflammatory diseases. There are numerous preparations of corticosteroids including oral tablets, capsules, liquids, topical creams and gels, inhalers, eye drops, and injectable and intravenous solutions.
  • Methylprednisolone is typically prescribed as an oral tablet or liquid.
  • Methylprednisolone is used to achieve prompt suppression of inflammation.
  • inflammatory conditions for which methylprednisolone is used include rheumatoid arthritis, systemic lupus erythmatosus, acute gouty arthritis, psoriatic arthritis, ulcerative colitis, and Crohn's disease.
  • Severe allergic conditions that fail conventional treatment also may respond to methylprednisolone. Examples include bronchial asthma, allergic rhinitis, drug-induced dermatitis, and contact and atopic dermatitis.
  • Chronic skin conditions treated with methylprednisolone include dermatitis herpetiformis, pemphigus, severe psoriasis and severe seborrheic dermatitis.
  • Chronic allergic and inflammatory conditions of the uvea, iris, conjunctiva and optic nerves of the eyes also are treated with methylprednisolone.
  • Another aspect relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a combination according to the invention and a pharmaceutically acceptable carrier, diluent or excipient.
  • Another aspect of the invention relates to a pharmaceutical product comprising an inhibitor of CDK2 and/or CDK7 and/or CDK9 as described above, or a pharmaceutically acceptable salt thereof, and methylprednisolone as a combined preparation for simultaneous, sequential or separate use in therapy.
  • the inhibitor of CDK2 and/or CDK7 and/or CDK9 and methylprednisolone may be administered simultaneously, in combination, sequentially or separately (as part of a dosing regime).
  • “simultaneously” is used to mean that the two agents are administered concurrently, whereas the term “in combination” is used to mean they are administered, if not simultaneously, then “sequentially” within a timeframe that they both are available to act therapeutically within the same time-frame.
  • administration “sequentially” may permit one agent to be administered within 5 minutes, 10 minutes or a matter of hours after the other provided the circulatory half-life of the first administered agent is such that they are both concurrently present in therapeutically effective amounts.
  • the time delay between administration of the components will vary depending on the exact nature of the components, the interaction therebetween, and their respective half-lives.
  • composition comprising:
  • a further aspect relates to a combination according to the invention in the preparation of a medicament for treating a disease associated with antinuclear antibodies.
  • a further aspect of the invention relates to the use of an inhibitor of CDK2 and/or CDK7 and/or CDK9 as described above, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating drug-induced lupus, SLE wherein the medicament is for use in combination with methylprednisolone.
  • Yet another aspect of the invention relates to the use of methylprednisolone in the preparation of a medicament for treating drug-induced lupus, or SLE wherein the medicament is for use in combination with an inhibitor of CDK2 and/or CDK7 and/or CDK9 as described above, or a pharmaceutically acceptable salt thereof.
  • a further aspect of the invention relates to the use of an inhibitor of CDK2 and/or CDK7 and/or CDK9 as described above, or a pharmaceutically acceptable salt thereof, and methylprednisolone, in the preparation of a medicament for treating drug-induced lupus, or SLE.
  • Another aspect of the invention relates to the use of an inhibitor of CDK2 and/or CDK7 and/or CDK9 as described above, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of drug-induced lupus, or SLE wherein said treatment comprises administering to a subject simultaneously, sequentially or separately the inhibitor of CDK2 and/or CDK7 and/or CDK9, or a pharmaceutically acceptable salt thereof, and methylprednisolone.
  • the inhibitor of CDK2 and/or CDK7 and/or CDK9 and methylprednisolone are administered simultaneously or sequentially.
  • the inhibitor of CDK2 and/or CDK7 and/or CDK9 and methylprednisolone are administered simultaneously.
  • the inhibitor of CDK2 and/or CDK7 and/or CDK9 is administered to the subject prior to sequentially or separately administering methylprednisolone to said subject.
  • methylprednisolone is administered to the subject prior to sequentially or separately administering the inhibitor of CDK2 and/or CDK7 and/or CDK9 to said subject.
  • the inhibitor of CDK2 and/or CDK7 and/or CDK9 and methylprednisolone are administered sequentially.
  • the inhibitor of CDK2 and/or CDK7 and/or CDK9 and methylprednisolone are each administered in a therapeutically effective amount with respect to the individual components.
  • the inhibitor of CDK2 and/or CDK7 and/or CDK9 and methylprednisolone are each administered in a subtherapeutic amount with respect to the individual components.
  • the inhibitor of CDK2, CDK7 or CDK9 is as described above for the first aspect of the invention.
  • compositions relate to pharmaceutical compositions.
  • the compounds of the present invention can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy.
  • a pharmaceutical carrier excipient or diluent
  • the pharmaceutical compositions may be for human or animal usage in human and veterinary medicine.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985 ).
  • suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like.
  • suitable diluents include ethanol, glycerol and water.
  • compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • Suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
  • Suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
  • preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents may be also used.
  • the compounds used in the invention can be present as salts or esters, in particular pharmaceutically acceptable salts or esters.
  • salts of the compounds of the invention include suitable acid addition or base salts thereof.
  • suitable pharmaceutical salts may be found in Berge et al, J Pharm Sci, 66, 1-19 (1977 ). Salts are formed, for example with strong inorganic acids such as mineral acids, e.g.
  • sulphuric acid, phosphoric acid or hydrohalic acids with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C 1 -C 4 )-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid.
  • Esters are formed either using organic acids or alcohols/hydroxides, depending on the functional group being esterified.
  • Organic acids include carboxylic acids, such as alkanecarboxylic acids of 1 to 12 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acid, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C 1 -C 4 )-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-to
  • Suitable hydroxides include inorganic hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide.
  • Alcohols include alkanealcohols of 1-12 carbon atoms which may be unsubstituted or substituted, e.g. by a halogen).
  • the invention includes the use of, where appropriate, all enantiomers and tautomers of the compounds involved.
  • the man skilled in the art will recognise compounds that possess an optical properties (one or more chiral carbon atoms) or tautomeric characteristics.
  • the corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art.
  • the compounds used in the invention may exist as stereoisomers and/or geometric isomers - e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms.
  • the present invention contemplates the use of all the individual stereoisomers and geometric isomers of those agents, and mixtures thereof.
  • the terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).
  • the present invention also includes the use of all suitable isotopic variations of the agent or pharmaceutically acceptable salt thereof.
  • An isotopic variation of an agent of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature.
  • isotopes that can be incorporated into the agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F and 36 Cl, respectively.
  • isotopic variations of the agent and pharmaceutically acceptable salts thereof are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3 H, and carbon-14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2 H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.
  • the present invention also includes the use of solvate forms of the compounds of the present invention.
  • the terms used in the claims encompass these forms.
  • the invention furthermore relates to the use of compounds of the present invention in their various crystalline forms, polymorphic forms and (an)hydrous forms. It is well established within the pharmaceutical industry that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation form the solvents used in the synthetic preparation of such compounds.
  • the invention further includes the ues of compounds of the present invention in prodrug form.
  • prodrugs are generally compounds wherein one or more appropriate groups have been modified such that the modification may be reversed upon administration to a human or mammalian subject.
  • Such reversion is usually performed by an enzyme naturally present in such subject, though it is possible for a second agent to be administered together with such a prodrug in order to perform the reversion in vivo.
  • Examples of such modifications include ester (for example, any of those described above), wherein the reversion may be carried out be an esterase etc.
  • Other such systems will be well known to those skilled in the art.
  • compositions of the present invention may be adapted for oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration.
  • compositions for oral administration, particular use is made of compressed Tablets, pills, Tablets, gellules, drops, and capsules.
  • these compositions contain from 1 mg to 5000 mg and more preferably from 10 mg - 3000 mg, of active ingredient per dose.
  • compositions of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders.
  • the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin.
  • the active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.
  • Injectable forms may contain between 10-3000 mg, preferably between 10-1000 mg, of active ingredient per dose.
  • compositions may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.
  • a person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation.
  • a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.
  • the dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • the agent may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
  • one or more doses of 10 to 3500 mg/day will be administered to the patient.
  • Kinase activity was investigated by measuring the incorporation of radioactive phosphate from ATP into appropriate polypeptide substrates.
  • Recombinant protein kinases and kinase complexes were produced or obtained commercially.
  • Assays were performed using 96-well plates and appropriate assay buffers (typically 25 mM ⁇ -glycerophosphate, 20 mM MOPS, 5 mM EGTA, 1 mM DTT, 1 mM Na 3 VO 3 , pH 7.4), into which were added 2 - 4 ⁇ g of active enzyme with appropriate substrates.
  • the reactions were initiated by addition of Mg/ATP mix (15 mM MgCl 2 + 100 ⁇ M ATP with 30-50 kBq per well of [ ⁇ - 32 P]-ATP) and mixtures incubated as required at 30 °C. Reactions were stopped on ice, followed by filtration through p81 filterplates or GF/C filterplates (Whatman Polyfiltronics, Kent, UK). After washing 3 times with 75 mM aq orthophosphoric acid, plates were dried, scintillant added and incorporated radioactivity measured in a scintillation counter (TopCount, Packard Instruments, Pangbourne, Berks, UK).
  • CTD peptide substrate biotinyl-Ahx-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser) 4 -NH 2 ; 1 - 2 mg/mL
  • recombinant human CDK7/cyclin H, CDK9/cyclin T1, or CDK9/cyclin K (0.5 - 2 ⁇ g) were incubated for 45 min at 30 °C in the presence of varying amounts of test compound in 20 mM MOPS pH 7.2, 25mM ⁇ -glycerophosphate, 5 mM EGTA, 1 mM DTT, 1mM sodium vanadate, 15 mM MgCl 2 , and 100 ⁇ M ATP (containing a trace amount of 32 P ⁇ ATP) in a total volume of 25 ⁇ L in a 96-well microtiter plate.
  • the reaction was stopped by placing the plate on ice for 2 min. Avidin (50 ⁇ g) was added to each well, and the plate was incubated at room temp for 30 min. The samples were transferred to a 96-well P81 filter plate, and washed (4 x 200 ⁇ L per well) with 75 mM phosphoric acid. Microscint 40 scintillation liquid (50 ⁇ L) was added to each well, and the amount of 32 P incorporation for each sample was measured using a Packard Topcount microplate scintillation counter.
  • NZBxNZW F1 female mice (Harlan Italy s.r.1., Milano, Italy) of 2 months of age at the start of the experiment, were used. Animal care and treatment were conducted in accordance with the institutional guidelines that are in compliance with national (Decreto Legislativo n.116, Gazzetta Ufficiale suppl 40, 18 febbraio 1992, Circolare n.8, Gazzetta Ufficiale 14 luglio 1994) and international laws and policies ( EEC Council Directive 86/609, OJL358-1, December 1987 ; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, 1996 ). Animals were housed in a constant temperature room with a 12-hour dark/12-hour light cycle and were fed a standard diet.
  • NZBxNZW F1 mice were randomly allocated to the following groups:
  • Five normal CD-1 mice (Charles River Italia, Calco, Italy) were used as control.
  • mice were randomly allocated to the following groups:
  • Urinary protein concentration was determined by the Coomassie blue G dye-binding assay with bovine serum albumin as standard. Renal function was assessed as BUN in heparinized blood by the Reflotron test (Roche Diagnostics Corporation, Indianapolis, USA). BUN levels exceeding 30 mg/dl were considered abnormal (normal range in this laboratory for mice: 14-29 mg/dl).
  • the levels of anti-dsDNA autoantibodies were evaluated in the serum by an enzyme-immunoassay (Diastat anti-ds DNA kit, Bouty Laboratory, Milano, Italy) as described before ( Kidney Int, 53:726-734, 1998 ).
  • Serum levels of AST and ALT were measured using an autoanalyzer (CX5, Beckman Instruments Inc., Fullerton, CA).
  • At least 100 glomeruli were examined for each biopsy.
  • At least 10 fields per sample were examined at low magnification (10 x) for histologic scoring of the interstitium. All renal biopsies have been analyzed by the same pathologist, in a single-blind fashion.
  • Rat monoclonal antibody against a cytoplasmic antigen present in mouse monocytes and macrophages was used for the detection of infiltrating cells by immunoperoxidase technique. Sections were incubated for 30 minutes with 0.3% H2O2 in methanol to quench endogenous peroxidase. Then the tissue was permeabilized in 0.1% Triton X-100 in PBS 0.01 mol/L, PH 7.2, for 30 minutes and then incubated with normal goat serum (Vector Laboratories) for 30 minutes.
  • mice gained weight during the study. No difference in body weight was observed among the experimental groups. Food (Table 2) and water (Table 3) intake evaluated every two weeks from 2 to 5 months were comparable among vehicle and CYC202 treated mice.
  • the percentage of survival was not different from that recorded in vehicle-mice.
  • mice with heavy proteinuria were evaluated at different stages of the disease in all of the experimental groups. As shown in Table 5, in the vehicle group the percentage of mice with proteinuria progressively increased over time ( Figure 2 ). At the end of the study the percentage of proteinuric mice was 85%.
  • CYC202 given as a preventive therapy significantly delayed the onset of proteinuria compared to vehicle, in a dose-dependent manner (% proteinuric mice, month 8: 200 mg /kg, 23%, P ⁇ 0.01 vs vehicle; 100 mg/kg, 43%, P ⁇ 0.05 vs vehicle).
  • CYC202 was administered to lupus mice from 5 months of age a tendency toward a reduced percentage of proteinuric mice in respect to vehicle was observed, which however did not reach the statistical significance.
  • Renal function assessed by serum BUN was measured at 5 and 8 months. At 5 months serum BUN levels were within the normal range ( ⁇ 29 mg/dl) in all the experimental groups. In the vehicle group, renal function deteriorated with time and at 8 months 50% of the surviving animals had BUN levels ⁇ 30 mg/dl (Table 6). CYC202 given as preventive therapy resulted in a better renal function of lupus mice, whereas it was not effective when administered late.
  • mice given vehicle exhibited increasing levels of anti-DNA antibodies over time.
  • mice treated from 2 months with CYC202 at both doses showed anti-DNA antibody levels significantly lower than vehicle.
  • anti-DNA antibody concentration was numerically, albeit not significantly lower than vehicle at 8 months ( Figure 3 ).
  • Serum ALT and AST levels were measured in NZB/W F1 mice given 200 mg/kg CYC202 from 2 months or 100 mg/kg CYC202 from 5 months. Serum transaminase levels were not modified by treatments and values were similar to those of control mice (Table 8).
  • mice given vehicle revealed glomerular changes with intracapillary hypercellularity associated with a focal extracapillary proliferation.
  • Immune type of deposits were detected in the mesangium and on subendothelial aspect of the glomerular basement membrane. Tubular damage and interstitial inflammation were also observed.
  • Treatment from 2 months with CYC202 markedly limited glomerular hypercellularity, immune deposits, and tubulointerstitial damage. These effects were more pronounced when CYC202 was given at the dose of 200 mg/kg. Only a mild effect on renal morphology was observed in mice administered CYC202 from 5 months.
  • Kidneys were analysed for F4/80 positive monocytes/macrophages by immunohistochemical technique. A marked accumulation of F4/80 positive cells was present in the renal interstitium of NZB/W mice given vehicle (Table 10). Preventive treatment with 200 mg/kg CYC202 remarkably reduced the number of F4/80 positive monocytes/macrophages in respect to vehicle. CYC202 at the dose of 100 mg/kg limited albeit not to a statistically significant degree the interstitial accumulation of mononuclear cells. A numerical reduction of F4/80 positive cells was observed in the therapeutic study.
  • CYC202 (200 and 100 mg/kg) given as a preventive therapy from 2 months of age, retarded renal manifestation of lupus in NZB/W mice and remarkably prolonged life as compared to animals given vehicle.
  • CYC202 delayed the onset of proteinuria and renal function impairment, and limited glomerular and tubulointerstitial changes including the interstitial accumulation of mononuclear cells, the effects being more pronounced at the dose of 200 mg/kg.
  • a remarkable finding of the present study was the reduction of the levels of anti-DNA antibodies by CYC202, which could possibly be attributed to CYC202 effects on T cells which in turn may affect B cells.
  • CYC202 100 mg/kg
  • the administration of CYC202 initiated at 5 months of age resulted in a mild reduction of the percentage of proteinuric mice and of renal damage in respect to vehicle-mice. Survival was not ameliorated.
  • NZB/W F1 mice gained weight during the study. No difference in body weight was observed among the experimental groups.
  • Table 12 shows the cumulative percentage of mice with proteinuria >4 mg/day evaluated at different stages of the disease.
  • the association of CYC202 and MPS significantly delayed the onset of proteinuria compared to vehicle. In the interval from 7 to 10 months the proportion of proteinuric mice in the combined therapy group was significantly lower than in the vehicle group (6.2 to 43.8% versus 40 to 90%).
  • CYC202 or MPS administered as single therapies only partially affected the onset of proteinuria as compared to vehicle, with a significant reduction in the percentage of proteinuric mice being observed at 7.5 months for the CYC202 group.
  • Renal function assessed by serum BUN, was measured monthly from 5 (before treatment) to 12 months.
  • Table 13 shows the cumulative percentage of mice with BUN levels ⁇ 30 mg/dl. At 5 months serum BUN levels were within the normal range (i.e. 14 to 29 mg/dl) in all the experimental groups. In the vehicle group, renal function deteriorated with time. Thus, at 8 and 12 months 80% and 100% of animals, respectively, had BUN levels ⁇ 30 mg/dl. By contrast, only 27% and 53% of mice on the combined therapy had impaired renal function at these time points. CYC202 or MPS given alone displayed a less renoprotective effect than when used in combination.
  • Kidneys were analysed for F4/80 positive monocytes/macrophages by immunohistochemical technique. A marked accumulation of F4/80 positive cells was present in the renal interstitium of NZB/W mice given vehicle (Table 15). The combined administration of CYC202 and MPS resulted in 65% reduction of the number of monocytes/macrophages in respect to vehicle (P ⁇ 0.01). CYC202 given alone reduced interstitial infiltrates by 33%. In the group of mice treated with MPS alone interstitial accumulation of mononuclear cells was similar to that found in the vehicle group.
  • CYC202 combined with low dose methylprednisolone significantly prolonged the lupus mice lifespan.
  • the treatment was initiated at a phase of established disease, i.e. 5 months of age, when immune complex deposition is actively taking place.
  • the combined therapy delayed the onset of proteinuria, limited impairment of renal function and development of glomerular hypercellularity, immune deposits and tubulointerstitial changes.
  • An anti-inflammatory effect was also displayed, as indicated by a reduced accumulation of mononuclear cells into the renal interstitium.
  • the administration of CYC202 or methylprednisolone as single therapies resulted in a mild renoprotective effect.
  • T cell proliferation assay is able to provide a measure of the effect of a compound on antinuclear antibodies.
  • PBMC Peripheral blood mononuclear cell
  • PBMCs were seeded in 96 well plates at 1 ⁇ 10 5 cells/well, in 50 ⁇ L per well culture medium.
  • As assay control CCRF-CEM leukaemia and LP-1 multiple myeloma cells were seeded at 4000 and 5000 cells/well in 100 ⁇ L/well RPMI 1640 tissue culture medium/10% fetal calf serum (FCS), respectively, and grown without stimulation or compound treatment for the duration of the experiment. All stimulants and compounds were made up to 4 ⁇ final concentration in tissue culture medium and 25 ⁇ L of each drug and stimulant was then added to wells, for a final volume of 100 ⁇ L/well.
  • tissue culture medium was added to wells instead of stimulant and for untreated cells, 25 ⁇ L/well tissue culture medium containing equal volume DMSO (0.1 % final) was added. Three adjacent wells were treated for each stimulation/treatment condition and the mean value calculated for analysis.
  • Example 3 cells were allowed to settle for 2 hours before stimulating with either 50 ⁇ g/mL PHA (Phytohemagglutinin PHA-P, Sigma L9132) or 50/250 ng/mL PMA/I (Phorbol 12-myristate 13-acetate/Ionomycin, Sigma P8139/I0634).
  • PHA Physicalhemagglutinin PHA-P
  • PMA/I Platinum 12-myristate 13-acetate/Ionomycin, Sigma P8139/I0634
  • PBMCs were stimulated with ConA (Concanavalin A, Sigma C5275) at concentrations ranging from 0.5 ⁇ g/mL to 100 ⁇ g/mL to identify its stimulatory effect and optimal concentration.
  • ConA ConA
  • Example 4 PBMCs were stimulated immediately after seeding, with either 50 ⁇ g/mL as before or 10 ⁇ g/mL ConA.
  • Example 3 stimulated and unstimulated PBMCs were treated with compounds [1]-[10] at IC 50 , 2 ⁇ IC 50 , 3 ⁇ IC 50 and 4 ⁇ IC 50 concentrations. Compounds were added at two hours before stimulation (time of seeding), at the time of stimulation and two hours after stimulation and cells then incubated at 37°C, 5% CO 2 , for 50, 48 and 46 hours with compound, respectively, or until 48 hours after stimulation.
  • stimulated PBMCs were treated with the above compounds, plus compounds [11] and [12], with all compounds tested at 0.5 ⁇ IC 50 and 0.25 ⁇ IC 50 in addition to the previous concentrations. Compounds were added immediately after stimulation and cells incubated for 48 and 72 hours.
  • IC 50 s were based on the average 72 hour IC 50 s from in-house cytotoxicity assays on tumour cell panels (Table 16), except for compounds [1] and [3] for which IC 50 s were based on published IC 50 s previously used in-house, as no in-house IC 50 data was available.
  • Proliferation activity of PBMCs after stimulation and compound treatment was determined by measuring BrdU incorporation, using the Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche # 1 647 229).
  • BrdU colorimetric kit
  • This kit is a non-radioactive replacement method for 3 H thymidine incorporation.
  • Cells were labelled with BrdU for two hours prior to harvesting. Plates were then centrifuged for 10 minutes at 300g (RT), supernatant removed from wells by pipetting and cells dried for 1 hour at 60°C. FixDenat solution was added to plates (100 ⁇ L/well) and incubated for 30 minutes, then flicked to remove and anti-BrdU-POD solution added to plates (100 ⁇ L/well) and incubated for 90 minutes.
  • PBMCs Viability of PBMCs was assessed using Alamar Blue reagent (Biosource # DAL1100). Fresh 20% Alamar Blue was made up in culture medium and 100 ⁇ L added to each well for a 10% final concentration. Plates were incubated with Alamar Blue at 37°, 5% CO 2 , for 3 hours prior to harvesting time (48 or 72 hours) and fluorescence then read using excitation wavelength 535 nM and emission wavelength 595 nM, on a Tecan Ultra plate reader.
  • Example 3 concluded that all the compounds tested affect the proliferation activity of stimulated PBMC cells at concentrations as low as IC 50 .
  • a subsequent experiment (Example 4) was carried out to study the concentration dependence of this effect at drug concentrations lower than IC 50 . Treatments were repeated as before, with the addition of treatments at lower concentrations, 0.5 ⁇ and 0.25 ⁇ IC 50 , and DMSO control, for all compounds previously tested plus compounds [11] and [12]. Since the time of compound treatment relative to stimulation did not appear to be central for inhibition of proliferation activity, PBMCs were stimulated and compound treated at the same time, immediately after seeding. Cells were stimulated with either PHA as before or with 10 ⁇ g/mL ConA.
  • BrdU signals do not reduce to background levels with either stimulation, even at 4 ⁇ IC 50 . Furthermore, little correlation was seen between BrdU incorporation and compound concentration at the higher concentrations, consistent with our previous results. Without wishing to be bound by theory, this suggests that effect of compound [4] on proliferation activity is maximised at around 2 ⁇ IC 50 as for most of the other compounds, but the degree of inhibition achieved is lower.
  • Viability assessment by Alamar Blue assay showed that viability of PBMCs is reduced slightly with stimulation, but is then generally comparable between compound-treated, DMSO-treated and untreated PBMCs. Furthermore, no reduction in Alamar Blue counts is associated with increasing compound concentrations, confirming that the decreasing BrdU incorporation is not caused by cytotoxicity effects of the compounds and is purely representative of inhibition of proliferation activity.

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Claims (26)

  1. Utilisation d'un inhibiteur purique de CDK2 et/ou CDK7 et/ou CDK9 choisi parmi
    Figure imgb0095
    Figure imgb0096
    Figure imgb0097
     ou un sel pharmaceutiquement acceptable de celui-ci, dans la préparation d'un médicament pour traiter le lupus médicamenteux ou le lupus érythémateux disséminé (SLE).
  2. Utilisation selon la revendication 1 dans laquelle le médicament est destiné à traiter le lupus érythémateux disséminé (SLE).
  3. Utilisation selon l'une quelconque des revendications précédentes dans laquelle le médicament est pour utilisation en thérapie d'association avec la méthylprednisolone.
  4. Utilisation selon l'une quelconque des revendications précédentes dans laquelle l'inhibiteur de CDK2 et/ou CDK7 et/ou CDK9 est en une quantité suffisante pour retarder l'apparition de la protéinurie et d'une altération de la fonction rénale.
  5. Utilisation selon l'une quelconque des revendications précédentes dans laquelle l'inhibiteur de CDK2 et/ou CDK7 et/ou CDK9 est en une quantité suffisante pour réduire les changements glomérulaires et tubulo-interstitiels.
  6. Combinaison comprenant un inhibiteur purique de CDK2 et/ou CDK7 et/ou CDK9, choisi parmi :
    Figure imgb0098
    Figure imgb0099
    Figure imgb0100
     ou un sel pharmaceutiquement acceptable de celui-ci, et de la méthylprednisolone.
  7. Composition pharmaceutique comprenant une combinaison selon la revendication 6 et un véhicule, diluant ou excipient pharmaceutiquement acceptable.
  8. Produit pharmaceutique comprenant un inhibiteur purique de CDK2 et/ou CDK7 et/ou CDK9 choisi parmi
    Figure imgb0101
    Figure imgb0102
    Figure imgb0103
     ou un sel pharmaceutiquement acceptable de celui-ci, et de la méthylprednisolone sous la forme d'une préparation combinée pour utilisation simultanée, séquentielle ou séparée en thérapie.
  9. Produit pharmaceutique selon la revendication 8, l'inhibiteur purique de CDK2 et/ou CDK7 et/ou CDK9, ou un sel pharmaceutiquement acceptable de celui-ci, et la méthylprednisolone étant administrés simultanément.
  10. Produit pharmaceutique selon la revendication 8 ou la revendication 9 qui comprend en outre un véhicule, diluant ou excipient pharmaceutiquement acceptable.
  11. Produit pharmaceutique selon l'une quelconque des revendications 8 à 10 pour utilisation dans le traitement d'une maladie rhumatismale auto-immune ou une auto-immunité d'organe spécifique.
  12. Produit pharmaceutique selon la revendication 11 dans lequel la maladie rhumatismale auto-immune est choisie parmi le lupus médicamenteux ou le lupus érythémateux disséminé (SLE).
  13. Produit pharmaceutique selon la revendication 12 dans lequel la maladie rhumatismale auto-immune est le lupus érythémateux disséminé (SLE).
  14. Composition pharmaceutique comprenant :
    (i) un inhibiteur purique de CDK2 et/ou CDK7 et/ou CDK9 choisi parmi
    Figure imgb0104
    Figure imgb0105
    Figure imgb0106
     ou un sel pharmaceutiquement acceptable de celui-ci ; et
    (ii) de la méthylprednisolone ;
    mélangés avec un diluant, excipient ou véhicule pharmaceutiquement acceptable.
  15. Composition pharmaceutique selon la revendication 7 ou 14, ou produit pharmaceutique selon l'une quelconque des revendications 8 à 13, l'inhibiteur purique de CDK2 et/ou CDK7 et/ou CDK9 étant choisi parmi la roscovitine et l'olomoucine et des sels pharmaceutiquement acceptables de celles-ci.
  16. Composition pharmaceutique selon la revendication 7 ou 14, ou produit pharmaceutique selon l'une quelconque des revendications 8 à 13, l'inhibiteur purique de CDK2 et/ou CDK7 et/ou CDK9 étant la roscovitine, ou un sel pharmaceutiquement acceptable de celle-ci.
  17. Utilisation d'une combinaison selon la revendication 6 dans la préparation d'un médicament pour traiter le lupus médicamenteux, ou le lupus érythémateux disséminé (SLE).
  18. Utilisation d'un inhibiteur purique de CDK2 et/ou CDK7 et/ou CDK9 choisi parmi
    Figure imgb0107
    Figure imgb0108
    Figure imgb0109
     ou un sel pharmaceutiquement acceptable de celui-ci, dans la préparation d'un médicament pour traiter le lupus médicamenteux ou le lupus érythémateux disséminé (SLE), le médicament étant pour utilisation en combinaison avec la méthylprednisolone.
  19. Utilisation de méthylprednisolone dans la préparation d'un médicament pour traiter le lupus médicamenteux ou le lupus érythémateux disséminé (SLE), le médicament étant pour utilisation en combinaison avec un inhibiteur purique de CDK2 et/ou CDK7 et/ou CDK9 choisi parmi
    Figure imgb0110
    Figure imgb0111
    Figure imgb0112
     ou un sel pharmaceutiquement acceptable de celui-ci.
  20. Utilisation d'un inhibiteur purique de CDK2 et/ou CDK7 et/ou CDK9 choisi parmi
    Figure imgb0113
    Figure imgb0114
    Figure imgb0115
     ou un sel pharmaceutiquement acceptable de celui-ci, et de méthylprednisolone, dans la préparation d'un médicament pour traiter le lupus médicamenteux ou le lupus érythémateux disséminé (SLE).
  21. Utilisation d'un inhibiteur purique de CDK2 et/ou CDK7 et/ou CDK9 choisi parmi
    Figure imgb0116
    Figure imgb0117
    Figure imgb0118
     ou un sel pharmaceutiquement acceptable de celui-ci, dans la préparation d'un médicament pour le traitement du lupus médicamenteux ou du lupus érythémateux disséminé (SLE), ledit traitement comprenant l'administration à un sujet, simultanément, séquentiellement ou séparément de l'inhibiteur purique de CDK2 et/ou CDK7 et/ou CDK9, ou un sel pharmaceutiquement acceptable de celui-ci, et de méthylprednisolone.
  22. Utilisation selon l'une quelconque des revendications 17 à 21 dans laquelle la maladie rhumatismale auto-immune est le lupus érythémateux disséminé (SLE).
  23. Utilisation selon l'une quelconque des revendications 1 à 3 ou 17 à 22, dans laquelle l'inhibiteur purique de CDK2 et/ou CDK7 et/ou CDK9 est choisi parmi la roscovitine et l'olomoucine.
  24. Utilisation selon l'une quelconque des revendications 1 à 13 ou 17 à 22, dans laquelle l'inhibiteur purique de CDK2 et/ou CDK7 et/ou CDK9 est la roscovitine, ou un sel pharmaceutiquement acceptable de celle-ci.
  25. Inhibiteur purique de CDK2 et/ou CDK7 et/ou CDK9 choisi parmi
    Figure imgb0119
    Figure imgb0120
    Figure imgb0121
     ou un sel pharmaceutiquement acceptable de celui-ci, pour utilisation dans le traitement du lupus médicamenteux ou du lupus érythémateux disséminé.
  26. Inhibiteur purique de CDK2 et/ou CDK7 et/ou CDK9 choisi parmi
    Figure imgb0122
    Figure imgb0123
    Figure imgb0124
     ou un sel pharmaceutiquement acceptable de celui-ci et méthylprednisolone, pour utilisation dans le traitement du lupus médicamenteux ou du lupus érythémateux disséminé.
EP05775408A 2004-08-27 2005-08-25 Purines et pyrimidines inhibant des CDK et leur utilisation pour le traitement des maladies autoimmunes Not-in-force EP1799230B1 (fr)

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AU2005276231A1 (en) 2006-03-02
CA2576159A1 (fr) 2006-03-02
ES2403558T3 (es) 2013-05-20
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WO2006021803A2 (fr) 2006-03-02
EP1799230A2 (fr) 2007-06-27
US20080125404A1 (en) 2008-05-29
JP5117189B2 (ja) 2013-01-09
WO2006021803A3 (fr) 2006-04-20

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